The attachment of a single N-acetylglucosamine (glucNAc) residue through an -O- linkage to serine/threonine residues of proteins is a recently discovered form of protein glycosylation. This type of glycosylation is found only on cytosolic and nuclear proteins of mammalian cells. In contrast, the classical forms of O- or N-linked glycosylation consists of large oligosaccharide structures found on externally oriented plasma membrane proteins. O-linked glucNAc glycosylation is dynamic in that cells have enzymes to attach and remove the sugar residues in response to yet unknown second messengers. This system may be analogous to protein kinases and phosphatases which regulate the addition of phosphate to serine/threonine or tyrosine residues and are widely used as second messengers in cellular signalling. We searched for O-linked glucNAc glycosylation in platelets. The glucNAc residue can be specifically labelled with radioactive UDP-galactose by galactosyltransferase, an enzyme isolated from bovine milk. In lysates of resting platelets we were able to label four major proteins with molecular mass of 159, 122, 79 and 52 kDa. The carbohydrate attachment of these labeled proteins was found to be O- linked since it was sensitive to B-elimination (alkali treatment) and resistant to peptide N-glycosidase (pNgase) treatment. The total O-linked glucNAc glycosylation increased by 20% when platelets were activated with agonists such as thrombin and collagen suggesting the possible dynamic nature of O-linked glucNAc in platelets. The identities of these proteins and their function during platelet activation is currently under investigation. The significance of this project lies in identification of new biochemical pathways of platelet activation which could potentially be used to modify platelet reactiveness in vivo or in vitro.